Skip to main content
SpringerLink
Log in

EV-T synergizes with AZD5582 to overcome TRAIL resistance through concomitant suppression of cFLIP, MCL-1, and IAPs in hepatocarcinoma

  • Original Article
  • Published:
Journal of Molecular Medicine Aims and scope Submit manuscript

Abstract

Hepatocellular carcinoma (HCC) is an aggressive malignancy, and its effective treatment has been hampered by drug resistance. Extracellular vesicle (EV) delivery of TNF-related apoptosis-inducing ligand (TRAIL) (EV-T) was demonstrated to be superior to recombinant TRAIL (rTRAIL) for cancer treatment previously. And AZD5582, a potent antagonist of inhibitors of apoptosis proteins (IAPs) can potentiate apoptosis-based cancer therapies. However, the combination of EV-T and AZD5582 has never been examined for their possible apoptosis inducing synergism in cancers. In this study, we proposed and tested the combination of EV-T and AZD5582 as a potential novel therapy for effective treatment of HCC. Two HCC lines Huh7 and HepG2 that are both resistant to rTRAIL were examined. The results confirmed that AZD5582 and EV-T are synergistic for apoptosis induction in some cancer lines including Huh7 and HepG2 while sparing normal cells. More importantly, this study revealed that TRAIL sensitization by AZD5582 is mediated through the concomitant suppression of anti-apoptotic factors including cFLIP, MCL-1, and IAPs (XIAP, Survivin and cIAP-1). Particularly the downregulation of cFLIP and IAP's appeared to be essential and necessary for the synergism between AZD5582 and TRAIL. In vivo, we first time demonstrated that the combined therapy with low doses of AZD5582 and EV-Ts triggered drastically enhanced apoptosis leading to the complete eradication of Huh7 tumor development without any apparent adverse side effects examined. We thus have unraveled the important molecular mechanism underlying TRAIL sensitization by AZD5582, rationalizing the next development of a combination therapy with AZD5582 and EV-T for HCC treatment.

Key messages

  • It confirmed the TRAIL sensitization by AZD5582, a potent antagonist of IAPs in hepatocarcinoma.

  • It revealed that the sensitization is via the concomitant suppression of antiapoptotic factors including cFLIP, MCL-1, and IAPs.

  • The downregulation of cFLIP and IAPs like Survivin appeared to be essential and necessary for the synergism between AZD5582 and nanosomal TRAIL.

  • In vivo the combined therapy with AZD5582 and nanosomal TRAIL led to complete eradication of hepatocarcinoma tumors.

  • This study has rationalized the next development of a combination therapy with AZD5582 and nanosomal TRAIL for cancer treatment.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Availability of data and materials

Not applicable.

Abbreviations

HCC:

Hepatocellular carcinoma

EV:

Extracellular vesicle

EV-T:

TRAIL-expressing extracellular vesicle

SMAC:

Second mitochondria-derived activator of caspase

rTRAIL:

Recombinant TRAIL

CCK-8:

Cell Counting Kit-8

TRAIL:

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand

DR:

Death receptor

MSC:

Mesenchymal stem cell

FADD:

Fas-associated with death domain protein

DISC:

Death-inducing signaling complex

MFI:

Median fluorescence intensity

FBS:

Fetal bovine serum

CDK:

Cyclin-dependent kinases

MOI:

Multiplicity of infection

Caspase:

Cysteinyl aspartate specific proteinase

PBS:

Phosphate buffered saline

IHC:

Immuno histochemistry

H&E:

Hematoxylin-eosin staining

IAPs:

Inhibitors of apoptosis proteins

NTA:

Nanoparticle tracking analysis

References

  1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A et al (2021) Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Ca-Cancer J Clin 71:209–249

    Article  PubMed  Google Scholar 

  2. Sawada Y, Yoshikawa T, Nobuoka D, Shirakawa H, Kuronuma T, Motomura Y et al (2012) Phase I trial of a glypican-3-derived peptide vaccine for advanced hepatocellular carcinoma: immunologic evidence and potential for improving overall survival. Clin Cancer Res 18:3686–3696

    Article  CAS  PubMed  Google Scholar 

  3. Kole C, Charalampakis N, Tsakatikas S, Vailas M, Moris D, Gkotsis E et al (2020) Immunotherapy for hepatocellular carcinoma: a 2021 update. Cancers (Basel) 12

  4. Kim DW, Talati C, Kim R (2017) Hepatocellular carcinoma (HCC): beyond sorafenib-chemotherapy. J Gastrointest Oncol 8:256–265

    Article  PubMed  PubMed Central  Google Scholar 

  5. Giannini EG, Farinati F, Ciccarese F, Pecorelli A, Rapaccini GL, Di Marco M et al (2015) Prognosis of untreated hepatocellular carcinoma. Hepatology 61:184–190

    Article  PubMed  Google Scholar 

  6. Jiang W, Lu Z, He Y, Diasio RB (1997) Dihydropyrimidine dehydrogenase activity in hepatocellular carcinoma: implication in 5-fluorouracil-based chemotherapy. Clin Cancer Res 3:395–399

    CAS  PubMed  Google Scholar 

  7. Kato A, Miyazaki M, Ambiru S, Yoshitomi H, Ito H, Nakagawa K et al (2001) Multidrug resistance gene (MDR-1) expression as a useful prognostic factor in patients with human hepatocellular carcinoma after surgical resection. J Surg Oncol 78:110–115

    Article  CAS  PubMed  Google Scholar 

  8. Straub CS (2011) Targeting IAPs as an approach to anti-cancer therapy. Curr Top Med Chem 11:291–316

    Article  CAS  PubMed  Google Scholar 

  9. Llovet JM, Kelley RK, Villanueva A, Singal AG, Pikarsky E, Roayaie S et al (2021) Hepatocellular carcinoma Nat Rev Dis Primers 7:6

    Article  PubMed  Google Scholar 

  10. Tang W, Chen Z, Zhang W, Cheng Y, Zhang B, Wu F et al (2020) The mechanisms of sorafenib resistance in hepatocellular carcinoma: theoretical basis and therapeutic aspects. Signal Transduct Target Ther 5:87

    Article  PubMed  PubMed Central  Google Scholar 

  11. Pitti RM, Marsters SA, Ruppert S, Donahue CJ, Moore A, Ashkenazi A (1996) Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family. J Biol Chem 271:12687–12690

    Article  CAS  PubMed  Google Scholar 

  12. Walczak H, Miller RE, Ariail K, Gliniak B, Griffith TS, Kubin M et al (1999) Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo. Nat Med 5:157–163

    Article  CAS  PubMed  Google Scholar 

  13. Micheau O, Shirley S, Dufour F (2013) Death receptors as targets in cancer. Br J Pharmacol 169:1723–1744

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Thapa B, Kc R, Uludağ H (2020) TRAIL therapy and prospective developments for cancer treatment. J Control Release 326:335–349

    Article  CAS  PubMed  Google Scholar 

  15. Ciaćma K, Więckiewicz J, Kędracka-Krok S, Kurtyka M, Stec M, Siedlar M et al (2018) Secretion of tumoricidal human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by recombinant Lactococcus lactis: optimization of in vitro synthesis conditions. Microb Cell Fact 17:177

    Article  PubMed  PubMed Central  Google Scholar 

  16. Wiezorek J, Holland P, Graves J (2010) Death receptor agonists as a targeted therapy for cancer. Clin Cancer Res 16:1701–1708

    Article  CAS  PubMed  Google Scholar 

  17. Wong SHM, Kong WY, Fang CM, Loh HS, Chuah LH, Abdullah S et al (2019) The TRAIL to cancer therapy: Hindrances and potential solutions. Crit Rev Oncol Hematol 143:81–94

    Article  PubMed  Google Scholar 

  18. Dimberg LY, Anderson CK, Camidge R, Behbakht K, Thorburn A, Ford HL (2013) On the TRAIL to successful cancer therapy? Predicting and counteracting resistance against TRAIL-based therapeutics. Oncogene 32:1341–1350

    Article  CAS  PubMed  Google Scholar 

  19. Wang F, Lin J, Xu R (2014) The molecular mechanisms of TRAIL resistance in cancer cells: help in designing new drugs. Curr Pharm Des 20:6714–6722

    Article  CAS  PubMed  Google Scholar 

  20. von Karstedt S, Montinaro A, Walczak H (2017) Exploring the TRAILs less travelled: TRAIL in cancer biology and therapy. Nat Rev Cancer 17:352–366

    Article  Google Scholar 

  21. Loebinger MR, Sage EK, Davies D, Janes SM (2010) TRAIL-expressing mesenchymal stem cells kill the putative cancer stem cell population. Br J Pharmacol 103:1692–1697

    CAS  Google Scholar 

  22. Sage EK, Kolluri KK, McNulty K, Lourenco Sda S, Kalber TL, Ordidge KL et al (2014) Systemic but not topical TRAIL-expressing mesenchymal stem cells reduce tumour growth in malignant mesothelioma. Thorax 69:638–647

    Article  PubMed  Google Scholar 

  23. Spano C, Grisendi G, Golinelli G, Rossignoli F, Prapa M, Bestagno M et al (2019) Soluble TRAIL armed human msc as gene therapy for pancreatic cancer. Sci Rep 9:1788

    Article  PubMed  PubMed Central  Google Scholar 

  24. Yuan Z, Lourenco Sda S, Sage EK, Kolluri KK, Lowdell MW, Janes SM (2016) Cryopreservation of human mesenchymal stromal cells expressing TRAIL for human anti-cancer therapy. Cytotherapy 18:860–869

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Yuan Z, Kolluri KK, Gowers KH, Janes SM (2017) TRAIL delivery by MSC-derived extracellular vesicles is an effective anticancer therapy. J Extracell Vesicles 6:1265291

    Article  PubMed  PubMed Central  Google Scholar 

  26. Ke C, Hou H, Li J, Su K, Huang C, Lin Y et al (2020) Extracellular vesicle delivery of TRAIL eradicates resistant tumor growth in combination with CDK inhibition by dinaciclib. Cancers (Basel) 12

  27. Hou H, Su K, Huang C, Yuan Q, Li S, Sun J et al (2021) TRAIL-Armed ER nanosomes induce drastically enhanced apoptosis in resistant tumor in combination with the antagonist of IAPs (AZD5582). Adv Healthc Mater 10:e2100030

  28. Yuan Z, Kolluri KK, Sage EK, Gowers KH, Janes SM (2015) Mesenchymal stromal cell delivery of full-length tumor necrosis factor-related apoptosis-inducing ligand is superior to soluble type for cancer therapy. Cytotherapy 17:885–896

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Rao Q, Zuo B, Lu Z, Gao X, You A, Wu C et al (2016) Tumor-derived exosomes elicit tumor suppression in murine hepatocellular carcinoma models and humans in vitro. Hepatology 64:456–472

    Article  CAS  PubMed  Google Scholar 

  30. Hennessy EJ, Adam A, Aquila BM, Castriotta LM, Cook D, Hattersley M et al (2013) Discovery of a novel class of dimeric Smac mimetics as potent IAP antagonists resulting in a clinical candidate for the treatment of cancer (AZD5582). J Med Chem 56:9897–9919

    Article  CAS  PubMed  Google Scholar 

  31. Belhadj Z, He B, Deng H, Song S, Zhang H, Wang X et al (2020) A combined “eat me/don’t eat me” strategy based on extracellular vesicles for anticancer nanomedicine. J Extracell Vesicles 9:1806444

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. O’Connor L, Harris AW, Strasser A (2000) CD95 (Fas/APO-1) and p53 signal apoptosis independently in diverse cell types. Cancer Res 60:1217–1220

    CAS  PubMed  Google Scholar 

  33. Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, Marsters SA et al (1999) Safety and antitumor activity of recombinant soluble Apo2 ligand. J Clin Invest 104:155–162

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chen J, Yu X, Huang Y (2016) Inhibitory mechanisms of glabridin on tyrosinase. Spectrochim Acta, Part A 168:111–117

    Article  CAS  Google Scholar 

  35. Lemke J, von Karstedt S, Abd El Hay M, Conti A, Arce F, Montinaro A et al (2014) Selective CDK9 inhibition overcomes TRAIL resistance by concomitant suppression of cFlip and Mcl-1. Cell Death Differ 21:491–502

    Article  CAS  PubMed  Google Scholar 

  36. Polanski R, Vincent J, Polanska UM, Petreus T, Tang EK (2015) Caspase-8 activation by TRAIL monotherapy predicts responses to IAPi and TRAIL combination treatment in breast cancer cell lines. Cell Death Dis 6:e1893

  37. Baglio SR, Pegtel DM, Baldini N (2012) Mesenchymal stem cell secreted vesicles provide novel opportunities in (stem) cell-free therapy. Front Physiol 3:359

    Article  PubMed  PubMed Central  Google Scholar 

  38. Walker S, Busatto S, Pham A, Tian M, Suh A, Carson K et al (2019) Extracellular vesicle-based drug delivery systems for cancer treatment. Theranostics 9:8001–8017

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Armstrong JP, Holme MN, Stevens MM (2017) Re-engineering extracellular vesicles as smart nanoscale therapeutics. ACS Nano 11:69–83

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. El-Andaloussi S, Lee Y, Lakhal-Littleton S, Li J, Seow Y, Gardiner C et al (2012) Exosome-mediated delivery of siRNA in vitro and in vivo. Nat Protoc 7:2112–2126

    Article  CAS  PubMed  Google Scholar 

  41. Hamidi H, Ivaska J (2018) Every step of the way: integrins in cancer progression and metastasis. Nat Rev Cancer 18:533–548

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Funding

This work was supported by the Guangdong Provincial Talented Scholar Foundation (grant number 220418137) and the National Natural Science Foundation of China (grant number 82173850).

Author information

Author notes

  1. Kui Su, Qian Yuan and Huan Hou equally contributed to this work.

Authors and Affiliations

  1. School of Biomedical and Pharmaceutical Sciences, Guangdong University of Technology, Guangzhou, 510006, People’s Republic of China

    Kui Su, Qian Yuan, Huan Hou, Chaohong Huang, Shuyi Li, Jianwu Sun, Xin Yuan, Yue Lin, Xiaoping Liang, Zhiyun Du & Zhengqiang Yuan

  2. School of Pharmacy, Jinan University, Guangzhou, 510632, People’s Republic of China

    Changhong Ke

  3. Department of Plastic and Reconstructive Surgery, Guangdong Second Provincial General Hospital, Guangzhou, 510317, People’s Republic of China

    Yiqing Chen

  4. Department of Ultrasound, Institute of Ultrasound in Musculoskeletal Sports Medicine, Guangdong Second Provincial General Hospital, Guangzhou, 510317, People’s Republic of China

    Huijuan Xin

Authors
  1. Kui Su
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qian Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Huan Hou
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Changhong Ke
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chaohong Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shuyi Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jianwu Sun
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Xin Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yue Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yiqing Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Huijuan Xin
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Xiaoping Liang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Zhiyun Du
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Zhengqiang Yuan
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.S., Q.Y., and H.H. contributed equally to this work. Conceptualization, K.S. Q.Y., H.H., X.P.L, and Z.Q.Y.; methodology, K.S., H.H., C.H.K., C.H.H., Y.Q.C., W.J.S., H.J.X, and Q.Y.; formal analysis, Z.Q.Y., X.P.L., C.H.K., and H.H; investigation, K.S., H.H., C.H.K., Q.Y., S.Y.L., X.Y., and J.W.S; resources, Z.Q.Y., Z.Y.D., and X.P.L.; writing-original draft preparation, K.S., Q.Y., H.H., and Z.Q.Y.; funding Acquisition, Z.Q.Y.

Corresponding authors

Correspondence to Xiaoping Liang, Zhiyun Du or Zhengqiang Yuan.

Ethics declarations

Ethics approval and consent to participate

All procedures and protocols for in vivo study had been approved by the Animal Ethics Committee in South China University of Technology (Approval ID: 20201210022; Date: 22 October 2020). All animal model experiments were carried out in accordance with the ethical standards outlined in the Best Practice Guidelines on Publishing Ethics.

Consent for publication:

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (PPTX 39 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Su, K., Yuan, Q., Hou, H. et al. EV-T synergizes with AZD5582 to overcome TRAIL resistance through concomitant suppression of cFLIP, MCL-1, and IAPs in hepatocarcinoma. J Mol Med 100, 629–643 (2022). https://doi.org/10.1007/s00109-022-02180-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00109-022-02180-9

Keywords

Search

Navigation